Safety and immunogenicity of ETVAX®, an oral inactivated vaccine against enterotoxigenic Escherichia coli diarrhoea: a double-blinded, randomized, placebo-controlled trial amongst Finnish travellers to Benin, West Africa.

BACKGROUND: No licensed human vaccines are available against enterotoxigenic Escherichia coli (ETEC), a major diarrhoeal pathogen affecting children in low- and middle-income countries and foreign travellers alike. ETVAX®, a multivalent oral whole-cell vaccine containing four inactivated ETEC strains and the heat-labile enterotoxin B subunit (LTB), has proved promising in Phase 1 and Phase 1/ 2 studies. METHODS: We conducted a Phase 2b double-blinded, randomized, placebo-controlled trial amongst Finnish travellers to Benin, West Africa. This report presents study design and safety and immunogenicity data. Volunteers aged 18-65 years were randomized 1:1 to receive ETVAX® or placebo. They visited Benin for 12 days, provided stool and blood samples and completed adverse event (AE) forms. IgA and IgG antibodies to LTB and O78 lipopolysaccharide (LPS) were measured by electrochemiluminescence. RESULTS: The AEs did not differ significantly between vaccine (n = 374) and placebo (n = 375) recipients. Of the solicited AEs, loose stools/diarrhoea (26.7/25.9%) and stomach ache (23.0/20.0%) were reported most commonly. Of all possibly/probably vaccine-related AEs, the most frequent were gastrointestinal symptoms (54.0/48.8%) and nervous system disorders (20.3/25.1%). Serious AEs were recorded for 4.3/5.6%, all unlikely to be vaccine related. Amongst the ETVAX® recipients, LTB-specific IgA antibodies increased 22-fold. For the 370/372 vaccine/placebo recipients, the frequency of ≥2-fold increases against LTB was 81/2.4%, and against O78 LPS 69/2.7%. The majority of ETVAX® recipients (93%) responded to either LTB or O78. CONCLUSIONS: This Phase 2b trial is the largest on ETVAX® undertaken amongst travellers to date. ETVAX® showed an excellent safety profile and proved strongly immunogenic, which encourages the further development of this vaccine.

Short interruptions of TNF-inhibitor treatment can be associated with treatment failure in patients with immune-mediated diseases.

INTRODUCTION: The prevalence of immune-mediated diseases has increased in the past decades and despite the use of biological treatments all patients do not achieve remission. The aim of this study was to characterise the reasons for short interruptions during treatment with two commonly used TNF-inhibitors infliximab and adalimumab and to analyse the possible effects of the interruptions on immunisation and switching the treatment. MATERIAL AND METHODS: This case-control study was based on retrospective analyses of patient records and a questionnaire survey to clinicians. A total of 370 patients (194 immunised cases and 172 non-immunised controls, 4 excluded) were enrolled from eight hospitals around Finland. Eleven different diagnoses were represented, and the largest patient groups were those with inflammatory bowel or rheumatic diseases. RESULTS: Treatment interruptions were associated with immunisation in patients using infliximab (p < .001) or adalimumab (p < .000001). Patients with treatment interruptions were more likely to have been treated with more than one biological agent compared to those without treatment interruptions. This was particularly prominent among patients with a rheumatic disease (p < .00001). The most frequent reason for a treatment interruption among the cases was an infection, whereas among the control patients it was remission. The median length of one interruption was one month (interquartile range 1-3 months). CONCLUSION: Our results suggest that the interruptions of the treatment with TNF-inhibitors expose patients to immunisation and increase the need for drug switching. These findings stress the importance of careful judgement of the need for a short interruption in the biological treatment in clinical work, especially during non-severe infections.

Immunogenicity of subcutaneous TNF inhibitors and its clinical significance in real-life setting in patients with spondyloarthritis.

KEY MESSAGES: Considerable proportion of patients with SpA have been immunized to the subcutaneous anti-TNF drug they are using. Concomitant use of MTX protects from immunization, whereas SASP does not. Patients with SpA using subcutaneous anti-TNF drugs can benefit from monitoring of the drug trough levels. Immunization to biological drugs can lead to decreased efficacy and increased risk of adverse effects. The objective of this cross-sectional study was to assess the extent and significance of immunization to subcutaneous tumor necrosis factor (TNF) inhibitors in axial spondyloarthritis (axSpA) patients in real-life setting. A serum sample was taken 1-2 days before the next drug injection. Drug trough concentrations, anti-drug antibodies (ADAb) and TNF-blocking capacity were measured in 273 patients with axSpA using subcutaneous anti-TNF drugs. The clinical activity of SpA was assessed using the Bath AS Disease Activity Index (BASDAI) and the Maastricht AS Entheses Score (MASES). ADAb were found in 11% of the 273 patients: in 21/99 (21%) of patients who used adalimumab, in 0/83 (0%) of those who used etanercept, in 2/79 (3%) of those who used golimumab and in 6/12 (50%) of those who used certolizumab pegol. Use of methotrexate reduced the risk of formation of ADAb, whereas sulfasalazine did not. Presence of ADAb resulted in decreased drug concentration and reduced TNF-blocking capacity. However, low levels of ADAb had no effect on TNF-blocking capacity and did not correlate with disease activity. The drug trough levels were below the consensus target level in 36% of the patients. High BMI correlated with low drug trough concentration. Patients with low drug trough levels had higher disease activity. The presence of anti-drug antibodies was associated with reduced drug trough levels, and the patients with low drug trough levels had higher disease activity. The drug trough levels were below target level in significant proportion of patients and, thus, measuring the drug concentration and ADAb could help to optimize the treatment in SpA patients.

Benefit of measuring vedolizumab concentrations in inflammatory bowel disease patients in a real-world setting.

OBJECTIVES: We set out to determine the reasons for serum vedolizumab (VDZ) trough concentration (TC) measurements in inflammatory bowel disease (IBD) patients and to evaluate treatment modifications after therapeutic drug measurement (TDM). We also evaluated the effect of increased dosing on patients' response to VDZ therapy. METHODS: We performed a retrospective cohort study of IBD patients who received VDZ therapy at Helsinki University Hospital and whose VDZ levels were measured between June 2014 and December 2018. RESULTS: Altogether, 90 patients (32 Crohn's disease and 58 ulcerative colitis) and 141 VDZ TC measurements were included. 24.1% of measurements took place during induction and 75.9% during the maintenance phase. During induction, 64.7% reached the target TC >20µg/ml. During maintenance therapy, 82.2% of VDZ TCs were within or exceeded the suggested target range of 5-15µg/ml. Reasons for TDM were: secondary nonresponse (44.0%), assessment of adequate VDZ TC (25.5%), primary nonresponse (12.8%), adverse events (6.4%), and other (11.3%). No treatment changes occurred after 60.3% of VDZ measurements. Increased dose frequency was used after 25.5% of VDZ measurements and 33.3% of these patients experienced improvement. Altogether, 31 (34.4%) patients discontinued the therapy due to inadequate treatment response. No anti-vedolizumab antibodies were detected. CONCLUSIONS: During the maintenance of VDZ therapy, the majority of VDZ TCs were within the suggested range. Measurement of VDZ TC did not lead to any treatment changes in two-thirds of patients. Dose optimization occurred in a quarter of patients and a third of them benefited from it.

Autoantibodies Against the Complement Regulator Factor H in the Serum of Patients With Neuromyelitis Optica Spectrum Disorder.

Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune inflammatory disease of the central nervous system (CNS), characterized by pathogenic, complement-activating autoantibodies against the main water channel in the CNS, aquaporin 4 (AQP4). NMOSD is frequently associated with additional autoantibodies and antibody-mediated diseases. Because the alternative pathway amplifies complement activation, our aim was to evaluate the presence of autoantibodies against the alternative pathway C3 convertase, its components C3b and factor B, and the complement regulator factor H (FH) in NMOSD. Four out of 45 AQP4-seropositive NMOSD patients (~9%) had FH autoantibodies in serum and none had antibodies to C3b, factor B and C3bBb. The FH autoantibody titers were low in three and high in one of the patients, and the avidity indexes were low. FH-IgG complexes were detected in the purified IgG fractions by Western blot. The autoantibodies bound to FH domains 19-20, and also recognized the homologous FH-related protein 1 (FHR-1), similar to FH autoantibodies associated with atypical hemolytic uremic syndrome (aHUS). However, in contrast to the majority of autoantibody-positive aHUS patients, these four NMOSD patients did not lack FHR-1. Analysis of autoantibody binding to FH19-20 mutants and linear synthetic peptides of the C-terminal FH and FHR-1 domains, as well as reduced FH, revealed differences in the exact binding sites of the autoantibodies. Importantly, all four autoantibodies inhibited C3b binding to FH. In conclusion, our results demonstrate that FH autoantibodies are not uncommon in NMOSD and suggest that generation of antibodies against complement regulating factors among other autoantibodies may contribute to the complement-mediated damage in NMOSD.

Assessing the Need for Multiplex and Multifunctional Tick-Borne Disease Test in Routine Clinical Laboratory Samples from Lyme Disease and Febrile Patients with a History of a Tick Bite.

Human polymicrobial infections in tick-borne disease (TBD) patients is an emerging public health theme. However, the requirement for holistic TBD tests in routine clinical laboratories is ambiguous. TICKPLEX® PLUS is a holistic TBD test utilized herein to assess the need for multiplex and multifunctional diagnostic tools in a routine clinical laboratory. The study involved 150 specimens categorized into Lyme disease (LD)-positive (n = 48), LD-negative (n = 30), and febrile patients from whom borrelia serology was requested (n = 72, later "febrile patients") based on reference test results from United Medix, Finland. Reference tests from DiaSorin, Immunetics, and Mikrogen Diagnostik followed the two-tier LD testing system. A comparison between the reference tests and TICKPLEX® PLUS produced 86%, 88%, and 87% positive, negative, and overall agreement, respectively. Additionally, up to 15% of LD and 11% of febrile patients responded to TBD related coinfections and opportunistic microbes. The results demonstrated that one (TICKPLEX® PLUS) test can aid in a LD diagnosis instead of four tests. Moreover, TBD is not limited to just LD, as the specimens produced immune responses to several TBD microbes. Lastly, the study indicated that the screening of febrile patients for TBDs could be a missed opportunity at reducing unreported patient cases.

Identifying the inheritable component of human thymic T cell repertoire generation in monozygous twins.

We have analyzed T cell receptor repertoires in a unique set of thymus samples from a pair of monozygotic twins. While genetics affect the V(D)J rearrangement and generation of junctional sequences, the thymic selections seem largely stochastic and import no detectable inheritable effect at clonal level.

Dientamoeba fragilis - the most common intestinal protozoan in the Helsinki Metropolitan Area, Finland, 2007 to 2017.

BackgroundDespite the global distribution of the intestinal protozoan Dientamoeba fragilis, its clinical picture remains unclear. This results from underdiagnosis: microscopic screening methods either lack sensitivity (wet preparation) or fail to reveal Dientamoeba (formalin-fixed sample).AimIn a retrospective study setting, we characterised the clinical picture of dientamoebiasis and compared it with giardiasis. In addition, we evaluated an improved approach to formalin-fixed samples for suitability in Dientamoeba diagnostics.MethodsThis study comprised four parts: (i) a descriptive part scrutinising rates of Dientamoeba findings; (ii) a methodological part analysing an approach to detect Dientamoeba-like structures in formalin samples; (iii) a clinical part comparing demographics and symptoms between patients with dientamoebiasis (n = 352) and giardiasis (n = 272), and (iv) a therapeutic part (n = 89 patients) investigating correlation between faecal eradication and clinical improvement.ResultsThe rate of Dientamoeba findings increased 20-fold after introducing criteria for Dientamoeba-like structures in formalin-fixed samples (88.9% sensitivity and 83.3% specificity). A further increase was seen after implementing faecal PCR. Compared with patients with giardiasis, the symptoms in the Dientamoeba group lasted longer and more often included abdominal pain, cramping, faecal urgency and loose rather than watery stools. Resolved symptoms correlated with successful faecal eradication (p < 0.001).ConclusionsPreviously underdiagnosed, Dientamoeba has become the most frequently recorded pathogenic enteroparasite in Finland. This presumably results from improved diagnostics with either PCR or detection of Dientamoeba-like structures in formalin-fixed samples, an approach applicable also in resource-poor settings. Symptoms of dientamoebiasis differ slightly from those of giardiasis; patients with distressing symptoms require treatment.

Role of Pneumococcal NanA Neuraminidase Activity in Peripheral Blood.

The most frequent form of hemolytic-uremic syndrome (HUS) is associated with infections caused by Shiga-like toxin-producing Enterohaemorrhagic Escherichia coli (STEC). In rarer cases HUS can be triggered by Streptococcus pneumoniae. While production of Shiga-like toxins explains STEC-HUS, the mechanisms of pneumococcal HUS are less well-known. S. pneumoniae produces neuraminidases with activity against cell surface sialic acids that are critical for factor H-mediated complement regulation on cells and platelets. The aim of this study was to find out whether S. pneumoniae neuraminidase NanA could trigger complement activation and hemolysis in whole blood. We studied clinical S. pneumoniae isolates and two laboratory strains, a wild-type strain expressing NanA, and a NanA deletion mutant for their ability to remove sialic acids from various human cells and platelets. Red blood cell lysis and activation of complement was measured ex vivo by incubating whole blood with bacterial culture supernatants. We show here that NanA expressing S. pneumoniae strains and isolates are able to remove sialic acids from cells, and platelets. Removal of sialic acids by NanA increased complement activity in whole blood, while absence of NanA blocked complement triggering and hemolytic activity indicating that removal of sialic acids by NanA could potentially trigger pHUS.

Chlamydia trachomatis samples testing falsely negative in the Aptima Combo 2 test in Finland, 2019.

Since February 2019, over 160 Chlamydia trachomatis (CT) cases testing negative or equivocal by Aptima Combo 2 (AC2) but positive by Aptima CT test run with Panther instruments occurred in Finland. The AC2 test targets chlamydial 23S rRNA while the CT test targets 16S rRNA. Sequencing of 10 strains revealed a nucleotide substitution in 23S rRNA. The significance of this for the failure of the AC2 test to detect the variant is not yet known.

Pool-seq driven proteogenomic database for Group G Streptococcus.

Proteogenomic databases use genomic and transcriptomic information for improved identification of peptides and proteins from mass spectrometry analyses. One application of such databases is in the discovery of variants/mutations. In this study, we created a proteogenomic database that contained sequences with variants derived from Pooled sequencing experiments (137 Group G Streptococcus strains sequenced in 3 pools) and used tandem mass spectrometry (MS/MS) to analyse eight protein samples from randomly selected strains sequenced in the pools. Using the proteogenomic variant database, we identified 385 variant peptides from the eight samples, none of which could be identified from the single genome conventional database utilized, while 71.2% and 93.5% of them were identified from the databases that contained 4 complete genomes and 26 assemblies, respectively. The proteogenomic variant databases exhibited the same properties as the conventional databases in terms of the Andromeda score distributions and the posterior error probability (PEP) values of the identified peptides. SIGNIFICANCE: For bacterial populations, such as Group G Streptococcus (GGS), with substantial intra-species diversity, simultaneous sequencing of large numbers of strains and generation of proteogenomic databases from those aids in improving the discovery of peptides in mass spectrometric analyses. Therefore, generation of proteogenomic variant protein databases from Pooled sequencing experiments can be a cost-effective method to complement conventional databases and discover subtle strain wise differences.

Complement Factor H and Apolipoprotein E Participate in Regulation of Inflammation in THP-1 Macrophages.

The alternative pathway (AP) of complement is constantly active in plasma and can easily be activated on self surfaces and trigger local inflammation. Host cells are protected from AP attack by Factor H (FH), the main AP regulator in plasma. Although complement is known to play a role in atherosclerosis, the mechanisms of its contribution are not fully understood. Since FH via its domains 5-7 binds apoliporotein E (apoE) and macrophages produce apoE we examined how FH could be involved in the antiatherogenic effects of apoE. We used blood peripheral monocytes and THP-1 monocyte/macrophage cells which were also loaded with acetylated low-density lipoprotein (LDL) to form foam cells. Binding of FH and apoE on these cells was analyzed by flow cytometry. High-density lipoprotein (HDL)-mediated cholesterol efflux of activated THP-1 cells was measured and transcriptomes of THP-1 cells using mRNA sequencing were determined. We found that binding of FH to human blood monocytes and cholesterol-loaded THP-1 macrophages increased apoE binding to these cells. Preincubation of fluorescent cholesterol labeled THP-1 macrophages in the presence of FH increased cholesterol efflux and cholesterol-loaded macrophages displayed reduced transcription of proinflammatory/proatherogenic factors and increased transcription of anti-inflammatory/anti-atherogenic factors. Further incubation of THP-1 cells with serum reduced C3b/iC3b deposition. Overall, our data indicate that apoE and FH interact with monocytic cells in a concerted action and this interaction reduces complement activation and inflammation in the atherosclerotic lesions. By this way FH may participate in mediating the beneficial effects of apoE in suppressing atherosclerotic lesion progression.

The Psoriasis Risk Allele HLA-C*06:02 Shows Evidence of Association with Chronic or Recurrent Streptococcal Tonsillitis.

Pharyngeal tonsillitis is one of the most common upper respiratory tract infections, and group A streptococcus is the most important bacterial pathogen causing it. While most patients experience tonsillitis only rarely, a subset of patients suffers from recurrent or chronic tonsillitis or pharyngitis. The predisposing factors for recurring or chronic forms of this disease are not yet fully understood, but genetic predisposition has been suggested. A genetic association study using Illumina's Immunochip single-nucleotide polymorphism (SNP) array was performed to search for new genetic biomarkers in pharyngeal tonsillitis. More than 100,000 SNPs relevant to immune-mediated diseases were analyzed in a cohort of 95 patients subjected to tonsillectomy due to recurrent/chronic tonsillitis and 504 controls. Genetic association between the cases and controls showed strongest association with two peaks in the HLA locus (odds ratio [OR], 3.7 to 4.7; P = 4.9 × 10-6 to 5.7 × 10-6). Further analysis with imputed classical HLA alleles suggested the known psoriasis risk allele HLA-C*06:02 as a risk factor for tonsillitis (P = 4.8 × 10-4; OR, 2.3). In addition, the imputed HLA haplotype HLA-C*06:02/HLA-B*57:01, a reported risk haplotype in psoriasis, had the strongest risk for tonsillitis (P = 3.2 × 10-4; OR, 6.5). These findings further support the previously reported link between streptococcal throat infections and psoriasis.

Distribution of exogenous complement factor H in mice in vivo.

Factor H is an important regulator of complement activation in plasma and on cell surfaces in both humans and mice. If FH function is compromised, inappropriate complement activation on self-surfaces can have disastrous effects as seen in the kidney diseases atypical haemolytic uremic syndrome (aHUS) and C3 glomerulopathy. As FH constructs have been proposed to be used in treatment for these diseases, we studied the distribution of exogenous FH fragments in mice. Full-length mFH, mFH1-5 and mFH18-20 fragments were radiolabelled, and their distribution was examined in WT, FH-/- and FH-/- C3-/- mice in vivo. Whole body scintigraphy revealed accumulation of radioactivity in the abdominal part of the mice, but also to the thyroid gland and urinary bladder. At organ level in WT mice, some full-length FH accumulated in internal organs, but most of it remained in the circulation. Both of the mFH fragments accumulated in the kidneys and were excreted in urine. For mFH1-5, urinary secretion is the likely cause for the accumulation. Concentration of mFH18-20 to kidneys was slower, and at tissue level, mFH18-20 was localized at the proximal tubuli in WT and FH-/- C3-/- mice. No C3-independent binding to glomeruli was detected. In conclusion, these results show that glomerular glycosaminoglycans and sialic acids alone do not collect FH in kidneys. Deposition of C3 fragments is also needed, which implies that in aHUS, the problem is in simultaneous recognition of C3 fragments and glycosaminoglycans or sialic acids by FH, not just the inability of FH to recognize glomerular endothelium as such.

Crystal structure of a tripartite complex between C3dg, C-terminal domains of factor H and OspE of Borrelia burgdorferi.

Complement is an important part of innate immunity. The alternative pathway of complement is activated when the main opsonin, C3b coats non-protected surfaces leading to opsonisation, phagocytosis and cell lysis. The alternative pathway is tightly controlled to prevent autoactivation towards host cells. The main regulator of the alternative pathway is factor H (FH), a soluble glycoprotein that terminates complement activation in multiple ways. FH recognizes host cell surfaces via domains 19-20 (FH19-20). All microbes including Borrelia burgdorferi, the causative agent of Lyme borreliosis, must evade complement activation to allow the infectious agent to survive in its host. One major mechanism that Borrelia uses is to recruit FH from host. Several outer surface proteins (Osp) have been described to bind FH via the C-terminus, and OspE is one of them. Here we report the structure of the tripartite complex formed by OspE, FH19-20 and C3dg at 3.18 Å, showing that OspE and C3dg can bind simultaneously to FH19-20. This verifies that FH19-20 interacts via the "common microbial binding site" on domain 20 with OspE and simultaneously and independently via domain 19 with C3dg. The spatial organization of the tripartite complex explains how OspE on the bacterial surface binds FH19-20, leaving FH fully available to protect the bacteria against complement. Additionally, formation of tripartite complex between FH, microbial protein and C3dg might enable enhanced protection, particularly on those regions on the bacteria where previous complement activation led to deposition of C3d. This might be especially important for slow-growing bacteria that cause chronic disease like Borrelia burgdorferi.

Differential diagnosis of thrombotic microangiopathy in nephrology.

BACKGROUND: The differential diagnosis of thrombotic microangiopathy (TMA) is complex however the rapid diagnosis of the underlying condition is vital to inform urgent treatment decisions. A survey was devised with the objective of understanding current practices across Europe and the Middle East, and of challenges when diagnosing the cause of TMA. METHODS: Over 450 clinicians, from 16 countries were invited to complete an online survey. RESULTS: Of 254 respondents, the majority were nephrologists, had >10 years' experience in their specialty, and had diagnosed a patient with TMA. The triad of thrombocytopenia, haemolytic anaemia and acute kidney injury are the main diagnostic criteria used. Responses indicate that a differential diagnosis of TMA is usually made within 1-2 (53%) or 3-4 days (26%) of presentation. Similarly, therapy is usually initiated within the first 4 days (74%), however 13% report treatment initiation >1-week post-presentation. Extrarenal symptoms and a panoply of other conditions are considered when assessing the differential diagnosis of TMA. While 70 and 78% of respondents stated they always request complement protein levels and ADAMTS13 activity, respectively. Diagnostic considerations of paediatric and adult nephrologists varied. A greater proportion of paediatric than adult nephrologists consider extrarenal manifestations clinically related to a diagnosis of TMA; pulmonary (45% vs. 18%), gastrointestinal (67% vs. 50%), CNS (96% vs. 84%) and cardiovascular (54% vs. 42%), respectively. Variability in the availability of guidelines and extent of family history taken was also evident. CONCLUSIONS: This survey reveals the variability of current practices and the need for increased urgency among physicians in the differential diagnosis of TMA, despite their experience. Above all, the survey highlights the need for international clinical guidelines to provide systematically developed recommendations for understanding the relevance of complement protein levels, complement abnormalities and ADAMTS13 testing, in making a differential diagnosis of TMA. Such clinical guidelines would enable physicians to make a more rapid and informed diagnosis of TMA, therefore initiate effective treatment earlier, with a consequent improvement in patient outcomes.

FHR-1 Binds to C-Reactive Protein and Enhances Rather than Inhibits Complement Activation.

Factor H-related protein (FHR) 1 is one of the five human FHRs that share sequence and structural homology with the alternative pathway complement inhibitor FH. Genetic studies on disease associations and functional analyses indicate that FHR-1 enhances complement activation by competitive inhibition of FH binding to some surfaces and immune proteins. We have recently shown that FHR-1 binds to pentraxin 3. In this study, our aim was to investigate whether FHR-1 binds to another pentraxin, C-reactive protein (CRP), analyze the functional relevance of this interaction, and study the role of FHR-1 in complement activation and regulation. FHR-1 did not bind to native, pentameric CRP, but it bound strongly to monomeric CRP via its C-terminal domains. FHR-1 at high concentration competed with FH for CRP binding, indicating possible complement deregulation also on this ligand. FHR-1 did not inhibit regulation of solid-phase C3 convertase by FH and did not inhibit terminal complement complex formation induced by zymosan. On the contrary, by binding C3b, FHR-1 allowed C3 convertase formation and thereby enhanced complement activation. FHR-1/CRP interactions increased complement activation via the classical and alternative pathways on surfaces such as the extracellular matrix and necrotic cells. Altogether, these results identify CRP as a ligand for FHR-1 and suggest that FHR-1 enhances, rather than inhibits, complement activation, which may explain the protective effect of FHR-1 deficiency in age-related macular degeneration.

Sequence analysis of pooled bacterial samples enables identification of strain variation in group A streptococcus.

Knowledge of the genomic variation among different strains of a pathogenic microbial species can help in selecting optimal candidates for diagnostic assays and vaccine development. Pooled sequencing (Pool-seq) is a cost effective approach for population level genetic studies that require large numbers of samples such as various strains of a microbe. To test the use of Pool-seq in identifying variation, we pooled DNA of 100 Streptococcus pyogenes strains of different emm types in two pools, each containing 50 strains. We used four variant calling tools (Freebayes, UnifiedGenotyper, SNVer, and SAMtools) and one emm1 strain, SF370, as a reference genome. In total 63719 SNPs and 164 INDELs were identified in the two pools concordantly by at least two of the tools. Majority of the variants (93.4%) from six individually sequenced strains used in the pools could be identified from the two pools and 72.3% and 97.4% of the variants in the pools could be mined from the analysis of the 44 complete Str. pyogenes genomes and 3407 sequence runs deposited in the European Nucleotide Archive respectively. We conclude that DNA sequencing of pooled samples of large numbers of bacterial strains is a robust, rapid and cost-efficient way to discover sequence variation.

Analysis of Linear Antibody Epitopes on Factor H and CFHR1 Using Sera of Patients with Autoimmune Atypical Hemolytic Uremic Syndrome.

INTRODUCTION: In autoimmune atypical hemolytic uremic syndrome (aHUS), the complement regulator factor H (FH) is blocked by FH autoantibodies, while 90% of the patients carry a homozygous deletion of its homolog complement FH-related protein 1 (CFHR1). The functional consequence of FH-blockade is widely established; however, the molecular basis of autoantibody binding and the role of CFHR1 deficiency in disease pathogenesis are still unknown. We performed epitope mapping of FH to provide structural insight in the autoantibody recruitment on FH and potentially CFHR1. METHODS: Eight anti-FH positive aHUS patients were enrolled in this study. With overlapping synthetic FH and CFHR1 peptides, we located the amino acids (aa) involved in binding of acute and convalescence stage autoantibodies. We confirmed the location of the mapped epitopes using recombinant FH domains 19-20 that carried single-aa substitutions at the suspected antibody binding sites in three of our patients. Location of the linear epitopes and the introduced point mutations was visualized using crystal structures of the corresponding domains of FH and CFHR1. RESULTS: We identified three linear epitopes on FH (aa1157-1171; aa1177-1191; and aa1207-1226) and one on CFHR1 (aa276-290) that are recognized both in the acute and convalescence stages of aHUS. We observed a similar extent of autoantibody binding to the aHUS-specific epitope aa1177-1191 on FH and aa276-290 on CFHR1, despite seven of our patients being deficient for CFHR1. Epitope mapping with the domain constructs validated the location of the linear epitopes on FH with a distinct autoantibody binding motif within aa1183-1198 in line with published observations. SUMMARY: According to the results, the linear epitopes we identified are located close to each other on the crystal structure of FH domains 19-20. This tertiary configuration contains the amino acids reported to be involved in C3b and sialic acid binding on the regulator, which may explain the functional deficiency of FH in the presence of autoantibodies. The data we provide identify the exact structures involved in autoantibody recruitment on FH and confirm the presence of an autoantibody binding epitope on CFHR1.

HUS and atypical HUS.

Hemolytic uremic syndrome (HUS) is a thrombotic microangiopathy characterized by intravascular hemolysis, thrombocytopenia, and acute kidney failure. HUS is usually categorized as typical, caused by Shiga toxin-producing Escherichia coli (STEC) infection, as atypical HUS (aHUS), usually caused by uncontrolled complement activation, or as secondary HUS with a coexisting disease. In recent years, a general understanding of the pathogenetic mechanisms driving HUS has increased. Typical HUS (ie, STEC-HUS) follows a gastrointestinal infection with STEC, whereas aHUS is associated primarily with mutations or autoantibodies leading to dysregulated complement activation. Among the 30% to 50% of patients with HUS who have no detectable complement defect, some have either impaired diacylglycerol kinase ε (DGKε) activity, cobalamin C deficiency, or plasminogen deficiency. Some have secondary HUS with a coexisting disease or trigger such as autoimmunity, transplantation, cancer, infection, certain cytotoxic drugs, or pregnancy. The common pathogenetic features in STEC-HUS, aHUS, and secondary HUS are simultaneous damage to endothelial cells, intravascular hemolysis, and activation of platelets leading to a procoagulative state, formation of microthrombi, and tissue damage. In this review, the differences and similarities in the pathogenesis of STEC-HUS, aHUS, and secondary HUS are discussed. Common for the pathogenesis seems to be the vicious cycle of complement activation, endothelial cell damage, platelet activation, and thrombosis. This process can be stopped by therapeutic complement inhibition in most patients with aHUS, but usually not those with a DGKε mutation, and some patients with STEC-HUS or secondary HUS. Therefore, understanding the pathogenesis of the different forms of HUS may prove helpful in clinical practice.